Some physiologically active substances which are present in biological fluids (e.g. in blood) are balanced between a free form and a form bound to endogenous ligands present in the same fluids. For these substances, the free-portion level is generally known to be clinically more significant than the total-substance level. Only the free form is in fact responsible for biological activity. Observations made on both normal and pathologically afflicted humans confirmed that the variations observed in free-form concentrations more closely correlate to the patients' clinical condition than are those of the entire substance. This is particularly true of thyroid and steroid hormones (Robbins J. and Rall J. E., Recent Progr. Hormone Res., 13:161 (1957); Robbins J. and Rall J. E., Physiol. Rev., 40:415 (1960)).
Methods intended to measure the free portions of these hormones have long been known.
In the specific case of thyroxine (T4), early attempts to measure the free portion involved determining its total concentration, as described in U.S. Pat. No. 3,659,104, and then determining triiodothyronine (T3) uptake (U.S. Pat. No. 3,710,117), or effecting a complete T4 dissociation in the presence of labelled T4, followed by adsorption of the latter upon a resin and subsequent elution from the resin using a portion of the test serum (U.S. Pat. No. 3,941,564).
However, these methods can only provide a rough and often incorrect assessment of the free portion.
A need thus arose to find other methods in which the measured level is directly correlated to the free-analyte concentration or to its distribution between free and bound portions.
In principle, this objective can be achieved by introducing a new exogenous component and having it take part in the existing equilibrium between the free analyte form and the bound analyte form, which component should have the following characteristics:
a. be incapable of affecting substantially the free analyte fraction present in a sample of biological fluid; PA1 b. be capable of sequestering an analyte quantity directly correlated to the free portion; PA1 c. be easily separated from the other components which take part in the equilibrium. PA1 (2) "two step" methods which use the same exogenous ligand both to sequester and to measure an analyte do not have the required sensitivity; PA1 (3) "two step" methods (which use different PA1 (a) a first exogenous ligand (L1) capable of sequestering an analyte quantity correlated to the free portion present in a biological fluid; PA1 (b) a dissociating agent (D) capable of dissociasing the sequestered analyte from the first exogenous ligand; PA1 (c) a labelled form (M) of the analyte to be determined; PA1 (d) a second exogenous ligand (L2) capable of proportionally binding both the dissociated analyte and the labelled analyte (M) even in the presence of the dissociating agent (D); PA1 (e) a set of standard solutions (SS) made up of sera containing known quantities (e.g., serial dilutions) of total analyte (free+bound) for which the free analyte concentrations are known. PA1 A--an analyte to be determined; PA1 L1--a first specific exogenous ligand, preferably bound to the surface of a stationary or dispersed solid phase; PA1 L2--a second specific exogenous ligand, preferably bound to the surface of a stationary or dispersed solid phase; PA1 M--a predetermined quantity of labelled analyte; PA1 D--an agent capable of dissociating the analyte A from the first exogenous ligand L1, without affecting the ability of the second exogenous ligand L2 to bind to said analyte and to the labelled analyte M; PA1 SS--a set of standards comprising, e.g., solutions or sera with known free-analyte concentrations or a set of standard concentration values (e.g., in table or graph form). PA1 12.times.75 mm "MAXYSORP" polystyrene test tubes (NUNC, Roskilde, Denmark] PA1 human thyroxine binding globulin (TBG) (Boehringer, Mannheim, Germany) PA1 monosodium phosphate, disodium phosphate, sodium azide, sodium chloride, TRIS (tris-(hydroxymethyl) aminomethane), potassium chloride, calcium chloride, magnesium sulphate, pure, analytical grade (Merck, Darmstad, Germany) PA1 Streptavidin from Streptomyces avidinii, N-succinimide ester of biotinyl-E-aminocaproic acid (SPA, Milan, Italy) PA1 ANSA (magnesium salt of 8-anilino-1-napththalenesulphonic acid), gelatin for microbiology, HEPES (4-(2-hydroxyethyl)-1-piperazinyl) ethanesulphonic acid), HEPES sodium salt, thyroxine (T4), 3,3', 5-triiodothyronine (T3) (SIGMA, St Louis, Missouri).
The U.S. Pat. No. 4,225,574 describes an assay method for the determination of free analytes in biological fluids which is based on these principles.
Said method calls for a sample to be subjected to a chromatographic adsorption process using Sephadex LH-20 columns. As soon as the free analyte present in the sample is adsorbed upon the resin, its concentration in the sample is promptly restored by dissociation of the analyte from the bound portion. This adsorption and dissociation process continues until a balanced state between adsorbed analyte and free analyte in solution is reached. The quantity of adsorbed analyte is proportionate to the free-analyte concentration, and it is therefore possible to find the latter by determining the adsorbed quantity by radioimmunoassay and dividing it by the proportionality constant. This method is difficult to perform for the same reasons as all "two-step" methods (see below).
A variation, which is described in Belgian Patent BE 878687, uses an analyte-specific antibody as a sequestering agent in place of a resin. When said antibody is introduced into a fluid, in a quantity incapable of substantially upsetting the free-analyte/bound-analyte equilibrium present in the sample, the number of antibody-binding sites which are occupied by the analyte is proportionate to the free-analyte concentration present. The number of free sites is measured after removing endogenous carrier proteins by washing and then adding a labelled analyte which enables the free-analyte concentration to be determined.
The main drawback of the Belgian patent technique is the necessity to use an antibody with a very high affinity constant for the analyte. The sensitivity of this type of assay does not change in relation to the quantity of antibody used, but depends exclusively on its affinity constant. For instance:to obtain a satisfactory sensitivity when measuring free T4 an antibody with an affinity constant higher than 5.times.10.sup.10 L/mol is required. On the other hand, if one wants to determine with acceptable sensitivity the concentration of free T3, which is present in blood in a lower concentration than T4, antibodies with an even higher affinity constant will be required.
The last two methods described are based essentially on a two-step sequential incubation: sequestration and then measurement.
The European Patent Application EP 26103 describes a method for assay of free analytes which tries to avoid this two-step sequential incubation by adding an appropriately labelled analyte derivative called "analogue" to the test sample at the same time as the analyte sequestering agent (analyte-specific antibody). If a particular analogue can be selected which is capable of binding to the sequestering agent but incapable of binding to the natural ligands present in biological fluids and is, therefore, only capable of competing with the available free-analyte moiety in binding to the antibody, the level of the antibody-bound signal will change only in proportion to the free-analyte concentration in the biological fluid.
In the method described in the European Patent Application EP 89806, which is similar to the preceding one, the specific ligand is labelled, instead of the analyte derivative. The analyte and its derivative compete for binding to the labelled specific ligand and the measured level of specific ligand bound to the analyte derivative is used to determine the free analyte concentration.
The methods, such as the one above, which use an analyte derivative (so called "one-step" methods) are extremely easy to carry out, which makes them more usable for laboratory routine than the methods in which the sequestering step is separate from the measuring step (so called "two-step" methods).
However, a serious drawback in the "one step" methods which use the "analogue" technique is the fact that it is not easy to select such a particular reagent, as it tends to bind to the natural ligands which are constantly present in biological fluids. When this occurs, the measured level is affected by the concentration of said ligands present in the sample.
Thus far it has not been possible to obtain a derivative which was thoroughly free from this drawback, the results obtained by these methods did not prove sufficiently reliable. The largest discrepancies occur in the cases where the quantity and quality (e.g., as a result of genetic alteration) of the natural ligands are far from normal (Refetoff, S. Endocr. Rev. 10:275, 1989; Waltz, M. R., J. Endocrinol. Invest. 13:343, 1990) or when substances are co-present that may affect the equilibria (between free and bound analyte) present in the biological fluids, such as for instance: nonesterified fatty acids, drugs used by patients under treatment and all the substances which bind in particular to albumin (Stockigt J. R. et al., Clin. Endocrinol., 15:313 (1981); Bayer M. F., Clin. Chim. Acta, 130:391, (1983); Beck-Peccoz P. et al., "Free T4 and free T3 measurement in patients with antiiodothyronine autoantibodies" in: Albertini A., Ekins R., Eds. "Free Hormone in Blood", 231: (1982), Amsterdam: Elsevier/North Holland).
In an attempt to avoid the above drawbacks, European Patent Application EP 155104 describes a method for free-analyte measurement in biological fluids, which uses, in addition to an analyte analogue, differential blocking agents which are intended to prevent or dissociate the bond between the analogue and natural ligands, in particular albumin. However, the addition of said differential blocking agents to the sample to be measured substantially affects the free-analyte/bound-analyte equilibrium in the sample itself, which distorts the measured levels.
Based on what has been previously described, in the present state of the art:
(1) "two step" methods are more reliable than "one step" methods owing to the difficulty of finding an appropriate analogue for "one-step" methods;
techniques for the sequestering step than for the measuring step of a free analyte) entail difficult sample manipulations and are not practical for use in routine laboratory analyses. Examples are dialysis or adsorption or ultrafiltration for sequestering and immunoassay for measuring.